Abstract Lysosomal-rich enterocytes (LREs) are a specialized population of enterocytes in posterior mid-intestine of zebrafish that exhibit high fluid-phase endocytosis activity and possess a large supranuclear lysosomal vacuole. These cells resemble premature enterocytes that are found in the ileum of pre-weaning mammals, which are distinct from mature enterocytes for their ability to efficiently uptake and process nutrients, particularly milk macromolecules. The importance of premature enterocytes in the utilization of nutrients during neonatal stage is reported through many studies; however, the cellular processes mediating protein absorption and digestion in these enterocytes remains poorly understood. My long-term goal is to understand the mechanism of protein uptake and utilization in these specialized intestinal enterocytes of neonatal mammals by studying an analogous population of enterocytes in zebrafish, LREs. A recent study from my lab has shown that the expression of the endosomal membrane protein Plasmolipin (Pllp) is enriched in LREs and that Pllp is critical for differentiation and endocytic function of LREs. Building upon our recent discovery of the crucial regulator of LRE function, I will explore the key cellular processes occurring in LREs. The use of zebrafish as a model system will be advantageous for this study because the optical transparency of zebrafish embryos will enable visualization of various cellular processes. Thus, using zebrafish as a model, I aim to uncover the endocytic pathways mediating protein internalization and trafficking in LREs (Aim1) and the regulators of lysosomal vacuole biogenesis and function in LREs (Aim2). The findings of the proposed study will elucidate the cellular processes mediating protein absorption and transport in LREs and provide a novel insight into the mechanism of lysosomal vacuole biogenesis and function in LREs. Additionally, our findings could ultimately contribute to the development of therapeutic strategies to reduce the incidence and severity of neonatal protein malnutrition.